Simple methods for synthesizing and purifying oligonucleotides are now in great demand due to the utility of synthetic oligonucleotides in a wide variety of molecular biological techniques. Until recently, the method of choice for synthesizing oligonucleotides was the beta-cyanoethyl phosphoramidite method. S. L. Beaucage and M. H. Caruthers, Tet. Let. 22: 1859 (1981). In the phosphoramidite procedure, the first nucleotide (monomer 1 ) is bound by its 3' hydroxyl to a solid matrix while its 5' hydroxyl remains available for binding. The synthesis of the first internucleotide link is carried out by mixing bound monomer 1 with a second nucleotide that has a reactive 3'diisopropyl phosphoramidite group on its 3' hydroxyl and a blocking group on its 5' hydroxyl (monomer 2). In the presence of a weak acid, coupling of monomer 1 and monomer 2 occurs as a phosphodiester with phosphorus in a trivalent state. This is oxidized giving a phosphotriester where the phosphorus is pentavalent. The protecting group is then removed from monomer 2 and the presence is repeated.
Oligonucleotide synthesis using nucleoside H-phosphonates has been reported (Hale et al., J. Chem. Soc., p. 3291 (1957); Sekine et al., Tet. Let., 20:1145 (1979); Garegg et al., Chemica Scripta, 26:59 (1986) but became practical only with the recent introduction of pivaloyl chloride (trimethyl acetyl chloride) as the condensing agent. There since have been reported of successful use of this method in both deoxyribonucleotide and ribonucleotide syntheses. Garegg et al., Tet. Let., 27:4051-4054 (1986); Froehler et al., Nucl. Acid. Res., 14:5399-5407 (1986) ; Garegg et al., Tet. Let., 27:4055-4058 (1986). The H-phosphonate method offers several advantages over the beat-cyanoetyl phosphoramidite method. The H-phosphonate method can be accomplished in a shorter cycle time and the 3' phosphonate monomers have greater stability than that of the corresponding 3'-phosphoramidiates. Finally, a simple reaction can be used to prepare backbone-modified DNA or RNA from the H-phosphonate synthesis product.
The H-phosphonate methods of Froehler et al. and Garegg et al., although adequate for small scale synthesis (i.e., less than 1 .mu.mole), are not practical on a large scale (e.g., 10-20 .mu.mole). The main reason it is not practical is that the methods reported by these groups require 20-30 equivalents of monomer per coupling reaction. At this rate, the monomer consumption costs represent approximately 60% of the oligonucleotide assembly cost.
In a recent publication, Gaffney et al. report an effort to scale up H-phosphonate oligonucleotide synthesis to the 10-20 .mu.mole range, while reducing the monomer equivalents consumed per coupling reaction. Gaffney et al., Tet. Let., 29:2619-2622 (1988). However, in synthesizing an 8-mer (consuming 1.53 equivalents of H-phosphonate) and a 26-mer (consuming 5.5 equivalents of H-phosphonate), Gaffney'group reported an average coupling yield of only 81% and 87%, respectively. Because of these relatively low coupling efficiency as compared with prior art methods, the authors found it necessary to employ a separate capping step using cyanoethyl H-phosphonate to prevent the elongation of truncated failed sequences in subsequent synthetic cycles. This extra step was necessary because the self-capping efficiency for pivaloyl chloride (the coupling reagent) was found to be too low. According to the method of Gaffney et al., which assumed a 94% coupling yield, the expected result of a 20-mer synthetic reaction would be a crude mixture consisting of 24% product (20-mer) and 76% short chains (e.g., 19-mers, 18-mers, etc.).
In sum, the presently available methods for oligonucleotide synthesis which have ben shown to produce relatively efficient coupling yields (i.e., greater than 97%) have not proven to be cost-effective in large scale reactions because these methods require the use of 20-30 equivalents of monomer per coupling reaction. In addition, the presently available methods which are more cost-effective in large scale reactions have proven unsatisfactory due to less efficient coupling yields.
A method of oligonucleotide synthesis which produces relatively efficient coupling yields and is cost-effective for large scale reactions would be very useful.